Many species of bacteria swim by means of filamentous organelles called flagella. Flagellar motility contributes to the virulence of many disease-causing bacteria and is important to the normal functioning of many species in the environment. The flagellum consists of a slender helical filament that functions as a propeller, turned by a membrane-embedded motor that obtains its energy from the membrane ion gradient. Motors can turn either clockwise (CW) or counter- clockwise (CCW). Regulated reversals in motor direction are the basis for directed movement in response to particular stimuli such as nutrients or repellents. The direction of rotation is controlled by a large multi-protein complex called the switch complex. In E. coli and a number of other species, function of the flagellar motor is additionally regulated by two other proteins: YcgR, which binds the second messenger cyclic-di-GMP and inhibits motor function during the conversion from the motile to the biofilm (surface-associated) state, and H-NS, a protein that functions in binding DNA and regulating gene expression in addition to regulating the motor. Both YcgR and H-NS have been shown to act at the switch complex. Goals of the present research are to understand the direction-reversing mechanism of the flagellar switch complex, and the mechanisms of motor regulation by YcgR and H-NS. Four aims are proposed. The first is to characterize the responses of the switch complex to the chemotactic signaling molecule CheY-phosphate. This will be accomplished using a combination of structural, biochemical, and advanced spectroscopic methods. The second aim is to elucidate the mechanisms of motor regulation by YcgR and H-NS. Interactions of these proteins with the motor will be examined using structural and biochemical methods, and effects on motor function will be studied using various measures of motor performance. Goals will be to understand how YcgR inhibits motility as cells begin entry into the biofilm state, which is a process important in many infections, and to clarify the logic underlying the dual functions of H-NS in both motility and gene regulation. A third aim is to extend studies of the switch complex, which have previously been carried out mainly in E. coli, to the gram-positive species B. subtilis. Gram-positive species are abundant in nature and of great importance in both medicine and ecology. Guided by information developed in the E. coli system, studies of the B. subtilis switch should proceed in a relatively efficient fashion and will greatly augment our understanding of motility in bacteria. The final aim is to begin development of a formal quantitative model of the flagellar switch. This will provide a framework for integrating the available structural and mechanistic information and for exploring the implications of the model in quantitative terms.